Our long-term objective is to define the mechanisms that allow some bacteria to modify their cell wall peptidoglycans (PGs) to become resistant to PG hydrolases, bacteriocins, and cell wall-targeted anti-microbials. In particular we are interested in FemABX-like immunity proteins, which modify PG cross bridges so that they become resistant to hydrolysis by bacteriolytic endopeptidases. In this project we will define the activity of the zoocin a immunity factor (Zif), a member of this family that is unique because it modifies PG without any apparent change in cross bridge composition. We will accomplish this through the following specific aims: 1) Define the site of action of zoocin A. Identification of the site of action of the enzyme will help identify the most likely target for Zif. First we will identify the products from the digestion of synthetic peptide substrates by the enzyme. Next, we will use HPLC/MS analysis of fragments generated from a zoocin A, mutanolysin digestion of purified PG of a zoocin A-negative, zoocin A-sensitive knockout mutant of Streptococcus equi subsp. zooepidemicus 4881, the zoocin A-producing organism, to confirm the bond or bonds hydrolyzed by zoocin A. 2) Determine the change in PG structure caused by Zif. Based on preliminary data, we hypothesize that Zif inserts D-alanine for L-alanine in PG cross bridges. First we will determine the ratio of D-alanine to L-alanine in PGs from zif+ and zif- strains. If our hypothesis is incorrect, comparison of HPLC/MS analyses of fragments from various digests of PGs from isogenic zif+ and zif- strains will be used to identify the change caused by Zif. 3) Determine if Zif forms complexes with other proteins. We will look for interactions between Zif and both MurM and MurN, which insert amino acids into streptococcal cross bridges during PG synthesis, and with other proteins using pulldowns. A bacterial two-hybrid system will also be used to look for interactions. Furthermore, Zif appears to be toxic in Staphylococcus aureus. Therefore we will determine if Zif forms complexes with FemA, FemB, or FemX, which insert amino acids into the staphylococcal cross bridges during PG synthesis, or with another FemABX-like immunity protein, Epr (also known as Lif), by pulldowns and with a bacterial two-hybrid system. The factors that are involved in cell wall synthesis are targets for a number of antibiotics, such as penicillins and cephalosporins, and alternative cell wall synthesis pathways are involved in resistance to these antimicrobial agents. This project will provide additional information regarding cell wall synthesis and degradation that may offer new targets for chemotherapeutic intervention. [unreadable] [unreadable] [unreadable]